Human biliverdin reductase (“hBVR”) is a small (296 residue) soluble protein with two distinct types of activities, the commonly known reductase activity and the more recently characterized kinase activity (reviewed in Kapitulnik et al., “Pleiotropic Functions of Biliverdin Reductase: Cellular Signaling and Generation of Cytoprotective and Cytotoxic Bilirubin,” Trends Pharmacol. Sci. 30:129-137 (2009)). As a reductase, hBVR is unique among all enzymes described to date in being a dual pH/cofactor-dependent catalyst for conversion of biliverdin-IXα to bilirubin-IXα (Maines et al., “Purification and Characterization of Human Biliverdin Reductase,” Arch. Biochem. Biophys. 300: 320-326 (1993), Hayes et al., “The Effect of pH on the Initial Rate Kinetics of the Dimeric Biliverdin-IXalpha Reductase From the Cyanobacterium Synechocystis PCC6803,” FEBS J. 276: 4414-4425 (2009)). Biliverdin, the substrate for hBVR, is a product of heme (Fe-protoporphyrin IX) oxidation by the HO enzymes, the stress-inducible HO-1 and the constitutive HO-2 (Mancuso et al., “Bilirubin: An Endogenous Scavenger of Nitric Oxide and Reactive Nitrogen Species,” Redox Rep. 11:207-213 (2006), Ryter et al., “Carbon Monoxide and Bilirubin: Potential Therapies for Pulmonary/Vascular Injury and Disease,” Am. J. Respir. Cell. Mol. Biol. 36:175-182 (2007)). Activation of hBVR is a key component of cellular defense mechanisms. In its capacity as a kinase, hBVR transfers phosphates to target substrate serine, threonine, and tyrosine residues and, hence, is a member of the rare family of dual-specificity kinases that have been described by Hunter T., “Signaling—2000 and Beyond,” Cell 100:113-127 (2000).
The primary structure of hBVR features, arguably, an unprecedented number of consensus signaling motifs (Kapitulnik et al., “Pleiotropic Functions of Biliverdin Reductase: Cellular Signaling and Generation of Cytoprotective and Cytotoxic Bilirubin,” Trends Pharmacol. Sci. 30:129-137 (2009)). The motifs, with demonstrated functionality include nuclear localization and nuclear export signals, high and low affinity MAPK docking sites, a C-Box (F162GFP) that is contained in the bulky ring motif (FXFXXF), and a D(δ)-Box (K275KRILXXLXL), respectively (Jacobs et al., “Multiple Docking Sites on Substrate Proteins Form a Modular System that Mediates Recognition by ERK MAP Kinase,” Genes Dev. 13:163-175 (1999), Karin M., “Inflammation-Activated Protein Kinases As Targets for Drug Development,” Proc. Am. Thorac. Soc. 2:386-390 (2005)). Those docking sites, as well as the H280CX10CC motif (CC-Box), are present in the C-terminal half of hBVR. The cysteine-rich motif is also present in C1 and C2/C2-like domains (Newton et al., “Protein Kinase C: A Paradigm for Regulation of Protein Function by Two Membrane-Targeting Modules,” Biochim. Biophys. Acta 1376:155-172 (1998)).
At multiple levels hBVR exerts input in transduction of insulin/stress—response signals; this includes activation of both conventional and atypical Protein Kinase-C (PKC) family members such as PKC-βII and PKC-ζ, respectively (Lerner-Marmarosh et al., “Regulation of TNF-alpha-Activated PKC-zeta Signaling By the Human Biliverdin Reductase: Identification of Activating and Inhibitory Domains of the Reductase,” FASEB J. 21:3949-3962 (2007), Maines M. D., “Biliverdin Reductase: PKC Interaction At the Cross-Talk of MAPK and PI3K Signaling Pathways,” Antioxid. Redox Signal. 9:2187-2195 (2007), Lerner-Marmarosh et al., “Human Biliverdin Reductase is an ERK Activator; hBVR is an ERK Nuclear Transporter and is Required for MAPK Signaling,” Proc. Natl. Acad. Sci. U.S.A. 105:6870-6875 (2008), Wegiel et al., “Cell Surface Biliverdin Reductase Mediates Biliverdin-Induced Anti-Inflammatory Effects Via PI3K and AKT,” J. Biol. Chem. 284:21369-21378 (2009)). PKC-δ is a member of the novel PKC family of serine/threonine kinases (Benes et al., “Modulation of PKC-delta Tyrosine Phosphorylation and Activity in Salivary and PC-12 Cells by Src Kinases,” Am. J. Physiol. Cell Physiol. 280:C1498-1510 (2001)) that is activated as the result of a conformational change induced by its cofactor, phorbolester/diacylglycerol that results in release of auto inhibition and exposure of the activation loop. Ca2+ and phorbolesters are activator cofactors for conventional PKCs, but PKC-δ is unique in that its cysteine-rich C2-like domain does not bind Ca2+.
hBVR and PKC-δ are universally expressed in tissues. In the cell, the type of stimuli that activate hBVR and PKC-δ extensively overlap. In addition to insulin, hBVR and PKC-δ have in common a varied list of extracellular stimuli such as TNF-α and reactive oxygen species (ROS) that are linked to cell survival, apoptosis and proliferation (Jackson et al., “The Enigmatic Protein Kinase C delta: Complex Roles in Cell Proliferation and Survival,” FASEB J. 18:627-636 (2004)). Activation of the two enzymes has been viewed in context of a variety of functions, with the most notable being pro-apoptosis (PKC) and anti-apoptosis (hBVR), respectively (Kapitulnik et al., “Pleiotropic Functions of Biliverdin Reductase: Cellular Signaling and Generation of Cytoprotective and Cytotoxic Bilirubin,” Trends Pharmacol. Sci. 30:129-137 (2009), Gschwendt, M., “Protein kinase C Delta,” Eur. J. Biochem. 259:555-564 (1999), Miralem et al., “Small Interference RNA-Mediated Gene Silencing of Human Biliverdin Reductase, But Not That of Heme Oxygenase-1, Attenuates Arsenite-Mediated Induction of the Oxygenase and Increases Apoptosis in 293A Kidney Cells,” J. Biol. Chem. 280:17084-17092 (2005), Stempka et al., “Requirements of Protein Kinase cdelta for Catalytic Function. Role of Glutamic Acid 500 and Autophosphorylation on Serine 643,” J. Biol. Chem. 274:8886-8892 (1999)).
PKC-δ phosphorylates S/T residues in specific motifs (Nishikawa et al., “Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes,” J. Biol. Chem. 272:952-960 (1997), Hanks et al., “The Protein Kinase Family: Conserved Features and Deduced Phylogeny of the Catalytic Domains,” Science 241:42-52 (1988)), that are found in hBVR, for example, RXXS/T in KRNRYLS230FHFKSGSL, SXR/KS21 that flanks the ATP-binding domain of hBVR, and S294 that flanks the CC-Box (Maines et al., “Human Biliverdin IXalpha Reductase is a Zinc-Metalloprotein. Characterization of Purified and Escherichia Coli Expressed Enzymes,” Eur. J. Biochem. 235:372-381 (1996)). Y228LSF is in one of the consensus SH-2 domain binding sites of hBVR, with the tyrosine residue being a substrate for IRK (Lerner-Marmarosh et al., “Human Biliverdin Reductase: A Member of the Insulin Receptor Substrate Family With Serine/Threonine/Tyrosine Kinase Activity,” Proc. Natl. Acad. Sci. U.S.A. 102:7109-7114 (2005)). Based on the noted similarities in hBVR and PKC-δ in phosphorylation motifs, upstream activators and downstream effector kinases, while also considering the opposing outcome of their activation to cell death and survival, it is possible these proteins have integrated and closely linked activities. It would be desirable, therefore, to identify whether BVR or, more particularly, peptide fragments of BVR are capable of modulating the activity of PKC-δ.
The present invention is directed to overcoming these and other deficiencies in the art.